Project summary ? RP3 Klebsiella pneumoniae (KP) and Pseudomonas aeruginosa (PA) are major causes of healthcare-associated Infections (HAI) including surgical site and wound infections, pneumonias, catheter based infections, urinary tract infections, and septicemia, both in the USA and worldwide. Importantly, previously successful antibiotic regimens for KP and PA are rapidly becoming ineffective due to the growing incidence of antimicrobial resistance (AMR), including to antibiotics of last resort such as polymixin/colistin, threatening a return to the pre-antibiotic era. As the clinico-epidemiological patterns for nosocomial infections with these pathogens are similar, a broad spectrum approach is warranted and would offer a potentially straightforward way to meaningfully reduce their combined incidence. Vaccine and prophylactic antibody approaches are unaffected by the evasion mechanisms mediating resistance to antibiotics, and thus represent a promising approach toward reducing the burden of AMR KP and PA infections. There are no available vaccines or antibody-based preventive measures for KP and PA however. Our overall goal is to develop vaccine and antibody-based countermeasures to prevent KP and PA HAIs. We propose here to continue assessment of a promising glycoconjugate vaccine for KP and PA developed under Department of Defense funding that is based on coupling of the four most common KP lipopolysaccharide-associated O polysaccharide (OPS) serotypes (60- 80% of clinical isolates worldwide) with the two types of PA flagellar major subunit proteins. We additionally propose to develop a novel antibody-based approach to prevent KP colonization of the intestine, a major risk factor for subsequent infection, by secretion of anti-KP fimbrial multi-specific single-chain antibody constructs from an orally ingested Saccharomyces boulardii probiotic. In Aim 1, we will determine whether immunization with the glycoconjugate generates immunity against relevant KP and PA clinical isolates in different challenge models approximating pneumonia, sepsis and wound infection. In Aim 2, we will assess protection in models of immunosenescence and immuno-compromise. Aims 3 and 4 will be focused on the development of a S. boulardii strain engineered to secrete a multi-specific single-chain (VHH) antibody construct targeting the KP fimbriae types important for intestinal attachment and colonization. These aims will assess whether conjugate immunization and/or administration with S. boulardii secreting anti-fimbrial VHHs can prevent intestinal colonization with KP. At the conclusion of this project, we expect to have generated important preclinical data to support advancement of these products to Phase 1 clinical studies.